About the Disease
Aspergillosis is a group of fungal diseases which include allergic bronchopulmonary aspergillosis (ABPA), aspergilloma, chronic necrotising aspergillosis, hypersensitivity pneumonitis and invasive aspergillosis. Aspergillus fumigatus, along with other less frequently reported species of Aspergillus such as A. flavus and A. niger, is the major causative fungus. Majority of the fungal allergies are due to Aspergillus species, with spores (conidia) and mycelia of the fungus as the infectious forms. A. fumigatus is an ubiquitous microbe and universal in distribution. Aspergillus species grow at temperatures of 15 to 53° C. in contrast to many fungi that do not grow above 35° C. Two major A. fumigatus induced diseases are an allergic form, Allergic bronchopulmonary aspergillosis (ABPA) and an invasive form, Invasive pulmonary aspergillosis. Pulmonary aspergillosis is a serious threat to those immunocompromised as a result of disease or therapy, and has been identified as a major cause of morbidity and mortality in asthmatic and cystic fibrosis patients. Ref: Pulmonary aspergillosis: clinical presentation, diagnosis and therapy. Daly P, Kavanagh K. Br J Biomed Sci 2001;58(3):197-205.
Allergic Bronchopulmonary Aspergillosis (ABPA)
The allergic form of the Aspergillus induced disease is named as Allergic bronchopulmonary aspergillosis (ABPA), which is an immunological disease and depicts the immune mechanisms similar to that of asthma. ABPA is often encountered in patients of bronchial asthma (15%, 16 out of 107), cystic fibrosis (7.8%, 191 out of 12,447), sinusitis (13%, 3 out of 23), rhinitis (5%, 3 out of 62). [Chetty et al, 1985; Mastella et al, 2000; Panchal et al, 1997; Grammer et al, 1986]. A study with 35 patients of ABPA showed that 12 (33%) of them were misdiagnosed as patients of pulmonary tuberculosis and were treated with various antitubercular drugs [Behera et al, 1994]. A conglomeration of intertwined Aspergillus hyphae matted together with fibrin, mucus and cellular debris, within a pulmonary cavity or ectatic bronchus, is termed as Aspergilloma. Patients of aspergilloma usually have an underlying pulmonary disease such as fibrocystic sarcoidosis, cavitary tuberculosis or histoplasmosis, bullous emphysema, or fibrotic lung disease.
Reference is made to S. Saxena et al., “Association of Polymorphisms in the Collagen Region of SP-A2 with increased levels of total IgE antibodies and Eosinophilia in Patients with Allergic Bronchopulmonary Aspergillosis”, J. Allergy Clin. Immunol., Vol. 11(5) 1001-1007 (May 2003).
Diagnosis
Aspergillosis of the lung does not show characteristic clinico-radiological features to permit the diagnosis and should be considered in the differential diagnosis of tuberculosis, pneumonia, bronchiectasis, lung abscess and bronchial asthma. Early diagnosis of ABPA is important for the following reasons: firstly, to prevent irreversible damage of the bronchi and the lungs. Bronchiectasis and bronchiolitis are known sequelae of the disease and if undiagnosed in early stages, may lead to pulmonary fibrosis and respiratory failure. ABPA may be the cause of recurrent pneumonias in children and may increase the severity of asthma in some patients [Chetty et al, 1985]. Association of ABPA in cystic fibrosis may worsen the course and prognosis [Mastella et al, 2000]. ABPA has also been found in multiple members of a family and a need has been felt for screening all the family members of a newly diagnosed case. Occurrence of ABPA is also known to be associated with pulmonary tuberculosis. It has been observed that both diseases show similar clinical symptoms that cause a diagnostic dilemma.
Disease Loci Identified Until Now and Their Associations
Infectious conidia of Aspergillus fumigatus are prevalent in the air and the population is exposed to them. However, occurrence of ABPA is limited to individuals with asthma, cystic fibrosis, atopic and other immunocompetent individuals. ABPA is a disease with immunological complexity. Genotype analysis of the T-cell clones (specific for the Asp f 1 antigen), isolated from ABPA patients, showed that most of them are restricted by HLA-DR molecules (90% of the ABPA patients showing a phenotype either HLA-DR2, HLA-DR5 or both) [Knutsen et al, 1994; Chauhan et al, 1994; Chauhan et al, 1996; Chauhan et al, 1997]. HLA-DR molecules DR2, DR5, and possibly DR4 or DR7 contribute to susceptibility while HLA-DQ2 contributes to resistance. Further, a combination of these genetic elements may determine the outcome of ABPA in patients with cystic fibrosis and asthma. Detailed genotype analysis of ABPA patients revealed that susceptibility to ABPA is also associated with alleles of HLA-DR2 and HLA-DR5. The presence of DR4 or DR7 alleles in non-DR2/5 patients with ABPA suggested that these alleles may be contributing factors to pathogenesis. Chauhan et al, 2000, reported a significantly high frequency of HLA-DQ2 in patients without ABPA (67.4%), compared with patients with ABPA (20.5%) and normal control subjects (37.7%), suggesting that these alleles may confer protection in the population without ABPA.
Mutations in cystic fibrosis transmembrane protein (CFTR) encoding gene of cystic fiborsis patients lead to defective synthesis and regulation of cystic fibrosis transmembrane protein. This protein is directly involved in the transportation of chloride ions. Such a defect in chloride transportation results in thick mucus secretion in these patients facilitating colonisation of pulmonary tract by other microbes. Presence of mutations in CFTR gene in ABPA patients suggest that there is some association of ABPA with cystic fibrosis [Miller et al, 1996]. In a recent study, the frequency of CFTR mutation carriers was observed to be significantly higher in ABPA patients (6 of 21 patients; 28.5%) than in control asthmatic subjects (2 of 43 subjects; 4.6%; p=0.01) [Marchand et al, 2001]. Hence, susceptibility and resistance to ABPA may be associated with certain genetic factors.
Role of Human Lung Surfactant Proteins in Aspergillosis
Pulmonary surfactant proteins, SP-A and SP-D, are immune molecules which can directly interact with pathogens and allergens, stimulate immune cells and manipulate cytokine and chemokine profiles during host's immune response. Therapeutic administration of SP-A in murine model of invasive pulmonary aspergillosis can rescue mice from death. Treating mice, having ABPA, can suppress IgE levels, eosinophilia, pulmonary cellular infiltration and cause a marked shift from a pathogenic Th2 to a protective Th1 cytokine profile. These results highlight the potential of SP-A as novel therapeutics for lung allergy and infection. Therefore, the SP-A locus make particularly good candidate to be screened for predisposition to pulmonary infectious disease.
The human SP-A gene locus consists of 2 highly homologous functional genes, SP-A1 (GenBank Ref: M30838) and SP-A2 (GenBank Ref: M68519), and a pseudogene located on human chromosome 10q22.2-q23.1. Both functional genes consist of 4 coding exons. Karinch and Floros, 1995, reported that 5′UT of SP-A genes contains 4 (A, B, C, D for SP-A1) or 3 (A, B, D for SP-A2) untranslated exons, three of which (A, B, D) vary in length, and one of which C is new; these exons are alternatively spliced and the major splice patterns as well as their relative frequency vary between the two genes (the major pattern for SP-A1 is AD′ [81%] and the major patterns for SP-A2 are ABD [44%] and ABD′ [49%]). According to GenBank information there are six exons in SP-A2, including two non-coding and four coding exons (M68519). Several alleles that differ by a single amino acid had been identified in each SP-A gene (Floros et al., 1996). The alleles of the SP-A1 gene are denoted as ‘6A(n),’ and those of the SP-A2 gene as ‘1A(n)’ (Floros and Hoover, 1998). In Finland, Ramet et al. (2000) found that certain SP-A1 alleles, 6A(2) and 6A(3), and a SP-A1/SP-A2 haplotype, 6A(2)/1A(0), were associated with respiratory distress syndrome (RDS; 267450). The 6A(2) allele was overrepresented and the 6A(3) allele was underrepresented in infants with RDS. These associations were particularly strong among small premature infants born at gestational age less than 32 weeks. Ramet et al. (2001) reported that the frequency of specific surfactant protein-A haplotypes and genotypes differs between children with recurrent otitis media compared with a control population in Finland.
The prior art is lacking in any method that associates the allelic variants of SP-A2 gene to the ABPA susceptibility. The prior art is also lacking in any study that correlates the substructure of SP-A2 with predisposition to the ABPA.
Elevated levels of A. fumigatus specific IgE and IgG antibodies detectable by various serodiagnostic techniques is one of the important diagnostic criteria for ABPA. However, unfamiliarity with the diagnostic tests and nonavailability of certain serologic tests at clinical laboratories compounds the difficulty for the clinician. Reference standards of Aspergillus antigens for immunodiagnosis are not available till today either with the world health organisation (WHO) or any international agencies. This is mainly due to the complex nature of antigens of Aspergillus species, which require multiple purification processes. This is the first demonstration that relates to the application of SNP's in human SP-A2 gene for use such as molecular diagnosis and prediction of an individual's disease susceptibility to ABPA or otherwise, and/or the genetic analysis of SP-A2 gene in Indian population. The novelty of the present invention is in providing a method for detecting and associating allelic variants of SP-A2 gene with the disease for prediction of an individual's predisposition to ABPA.